1. Field of the Invention
This invention relates generally to the field of introducing samples to a mass spectrometer and more specifically to an interface for introducing samples obtained from gel electropherograms to mass spectrometry devices for analysis, including analysis using laser desorption.
2. Prior Art
Mass spectrometry is the most specific detection method available for coupling to chromatographic separation methods, and provides a high level of sensitivity for most compounds. Coupling of mass spectrometry to gas chromatography and liquid chromatography is well known. Supercritical fluid chromatography and capillary zone electrophoresis (CZE) also both have been coupled to mass spectrometers.
Devices for the direct analysis of liquid streams by mass spectrometry have been increasingly evident in recent literature. In particular, the development of continuous flow fast atom bombardment (FAB) mass spectrometry has been an area of vigorous research. Analytical advantages of the flow FAB probe include lower chemical background noise (compared to conventional FAB Probes), reduced ion suppression effects for mixtures of samples with varying surface activities, and generally lower detection limits. Practical advantages include the fact that introducing samples via a flow stream greatly simplifies and quickens FAB measurements even for discrete samples. Continuous-flow fast atom bombardment interfaces for liquid chromatography have been widely adopted.
Several references in the literature describe the coupling of capillary zone electrophoresis (CZE) with mass spectrometry. This is a fundamentally different form of separation and is not considered relevant to the present invention. Two recent papers describe an indirect coupling based on separate extraction of samples from gel electropherograms and off-line analysis by fast atom bombardment mass spectrometry (FABMS): P. Camilleri et al , 3 Rap. Comm. Mass Spec., 346 (1989); 440 (1989). Camilleri et al. reported the extraction of samples from polyacrylamide gels, with subsequent analysis of the discrete samples by FABMS. However, the necessary sample preparation included an extended extraction of the gel with strongly acidic solvents and crushing of the gels after sample bands were excised from the gel.
Similarly, Duffin et al. (as reported in an abstract at a recent meeting) have extracted biological samples from within gels with the aid of an extended sonication, time and a series of eluting solvents, and have shown that FAB analysis provides good quality mass spectra of these extracts. Duffin's extraction apparently involved a slice of gel about 0.5 cc in volume placed in a test tube of extraction solvent. The gel and solvent were sonicated overnight and then evaporated to near dryness. The residue was taken up in a FAB solvent and the analysis of the residue was performed as if the sample was a discrete sample generated by any other means. Duffin's Process is a standard recovery procedure known to those skilled in the art and samples recovered from procedures similar to Duffin's also are used in unrelated art. The data of Duffin show, and it is generally agreed that relatively drastic means are required to release large biological samples from gel matrices in which they are encapsulated. Even high power laser desorption can be insufficient for extraction, although our own recent results suggest that conditions can be found that release intact molecular ions of nucleotides from dried agarose gels.
Previous mentions of planar chromatography coupled with mass spectrometry are our own, but none of these relate specifically to the present invention. (K. L. Busch, 6 Trends Anal. Chem., 95 (1987); M. S. Stanley et al., 200 Anal. Chem. Acta, 447 (1987); M. S. Stanley and K. L. Busch, 1 J. Planar Chrom. 135 (1988). Our previous work in coupling planar chromatography with mass spectrometry relate to combinations of mass spectrometry with high performance thin-layer chromatography, or to applications of electrophoresis with mass spectrometry that involve transfer of the samples to a secondary substrate. In this former work, there is no separate interface necessary, as the primary particle beam sputters material from the gel surface directly. A phase-transition matrix sometimes is involved in the former work, but there is no transfer of material out of the chromatographic medium through capillary transfer lines to the source of the mass spectrometer as the entire chromatogram is placed within the vacuum chamber of the mass spectrometer. The present invention, when compared to the former work, had several distinguishing factors, including the present invention's ability to extract the sample material directly from a PAGE, agarose, or other gel with a variety of solvents; concentration of the sample; removal of extraneous components in the system; and the transfer of the sample in a flow stream of solvent through a capillary line to the source of the mass spectrometer.
The methods used for recovering material from gel electropherograms depend mainly on the subsequent steps to be performed on the recovered analyte, such as sequencing or in situ reactions (immunoassay). The general problem of recovering DNA/RNA or proteins from gels lies in the physical barriers that the large molecules encounter. That is, the long strands of biopolymer are so well enmeshed inside of the gel that the molecules have to be coaxed rather strongly to be released. The gel itself generally is immune to the types of chemical attack that are sufficient to destroy the biopolymer itself.
The need to recover materials from gels is ongoing, as the most widely used separation method by the biological community is planar electrophoresis, with an extensive tradition and repertoire built over years of experience with the method applied to provide high resolution, multi-dimensional separations of complex biological mixtures. PAGE (polyacrylamide gel electrophoresis) and agarose gel electrophoresis are high capacity, high precision, and high dynamic range methods. Bioanalytical protocols are based explicitly on these methods, and have been optimized over twenty years of continuous use. New developments in CZE and its variants will complement, but certainly will not supplant, the methods of planar electrophoresis. Although many detection methods have been developed in conjunction with planar electrophoresis, to date no process or method has coupled mass spectrometry with that separation method.
Most proteins are separated by polyacrylamide gel electrophoresis (PAGE) (based on the molecular weight) or modified polyacrylamide gel isoelectric focusing (based on molecular charge). Both of the techniques can be used in tandem in a two-dimensional approach for maximum resolution. Polyacrylamide gels are made by polymerizing the monomer, acrylamide, into long strands, and then linking the strands together with a `cross-linker`, usually N,N'-methylene-bis-acrylamide (Bis). The relative proportions of these components will determine the separation characteristics of the gel. Isoelectric focusing is carried out in a PAGE gel that contains an immobilized pH gradient consisting of high molecular weight polyaminocarboxylic acids (ampholytes).
Other known methods for separating a desired material from a gel include direct extraction, electroblotting, electroelution, capillary blotting, sonication, and electrophoresis.
The direct extraction method involves cutting out the band of interest from the gel, mashing it and immersing it in a buffer solution of Tris ((tris-hydroxymethyl)- aminomethane), glycine and SDS. The mixture is shaken, after which it is filtered and the protein recovered by extraction. This method is highly unsatisfactory for large proteins (extremely low recovery) even under these extreme conditions due to the fact that diffusion of large proteins from within the complex gel network is an inefficient process.
Direct extraction methods also can be used for small nucleic acid strands. Agarose gels can be dissolved in 6M NaClO.sub.4 ; the solution is then filtered, extracted with appropriate solvents and the DNA is precipitated. Nucleic acids separated by PAGE gels can be extracted directly in a manner similar to that for proteins by mashing the gel, putting it into a medium of high ionic strength, such as ammonium acetate, which promotes diffusion of DNA out of the gel, SDS, and a magnesium salt to aid precipitation of DNA. Sample molecules dissolved in the aqueous gel extract are then precipitated with ethanol.
Electroblotting, the most common and satisfactory method of recovering proteins, involves transfer of the proteins from the gel onto another equally sized membrane, using an electric current to drive their migration in a manner similar to the original electrophoresis (Western blot), but in a perpendicular direction. Although there are many variations on this technique, it essentially involves making a sandwich of the gel and the transfer membrane (commonly nitrocellulose) between two layers of filter paper. This sandwich is then placed into a tank of transfer buffer solution and a low current is passed through the tank across the sandwich. The reason for performing an electroblot is that the proteins are now more accessible on the transfer membrane than they were in the gel. For instance, detection techniques are more sensitive and the proteins on the membrane can be reacted in situ, with antibodies or other agents.
One commonly used method of recovering a sample by electroelution uses a dialysis bag. The portion of gel containing the nucleic acid of interest is cut out and put into a dialysis bag filled with buffer. After the gel has sunk to the bottom of the bag, the excess buffer can be removed. The bag is then immersed in a shallow tank of buffer and electric current is passed through the bag. The nucleic acid is then electroeluted onto the wall of the bag. The polarity of the current is reversed for a short time to release the nucleic acid from the wall of the bag. The nucleic acid is thereby recovered and purified. A second commonly used method of electroelution is trough electroelution, which involves cutting a trough in the gel on the leading edge of the selected band. The trough is filled with buffer and electrophoresis continues, with the nucleic acid being moved into the trough. The buffer in the trough is withdrawn and replaced a few times until all of the nucleic acid is recovered in solution. A more ingenious method involves putting a dialysis membrane vertically in the trough and electroeluting the nucleic acid from the gel onto the membrane.
Nucleic Acids (DNA/RNA) and their components commonly are separated by agarose gel electrophoresis, although PAGE often is used to separate complementary strands of denatured DNA/RNA.
Capillary blotting of DNA/RNA was developed first by Southern (Southern blot); the geographical pun is continued in the derived name of a Western Blot. In a Western blot, the blotting proceeds by capillary Pressure. It should be noted that the Southern blot also works well for peptides and small proteins from PAGE gels.
In order to make biomolecule recovery easier, the structure of the gels used in electrophoresis may be changed, thus changing their chemical or physical properties. PAGE gels can be modified by using cross linkers alternative to bisacrylamide. Alternative cross-linkers include N,N'-(1,2 dihydroxyethylene) bisacrylamide (DHEBA), ethylene diacrylate (EDA), and N,N'-bisacrylcystamine (BAC). Once the cross-linking is disrupted, the gel can be solubilized and the biomolecules can more readily diffuse out. As with any of the methods described above, the time required, the recovery, and the capability for repetitive application to the many bands separated on a gel are, in general, unsatisfactory.
In sonication extraction of samples from electropheretic gels, a small piece of the gel is excised from the larger gel, mixed with solvent in a tube or small flask, and then sonicated in a standard ultrasonic bath for some period of time, usually hours, but sometimes . overnight. The sonication increases the rate of diffusion of the sample molecules from within the pores of the electropheretic gel out into the solvent added to the sample. The sonication does not by itself usually disrupt the cross-link structure of the gel. Extended sonication can, however, result in degredation of the sample molecules themselves.
Extraordinary resolution in separation is available with CZE, but it is still a relatively new technique, and is used for compounds of relatively low mass in comparison with the masses of those separated by gel electrophoresis. Various forms of gel (agarose or polyacrylamide) electrophoresis are utilized in virtually every biochemical laboratory for samples of molecular weights of several thousand to several hundred thousand daltons. The coupling of mass spectrometry with these gel electrophoretic techniques would benefit a great number of researchers searching for a more selective and information-rich detection method.